We developed a Sleeping Beauty (SB) transposon mediated hTERT gene delivery system for in vitro use. We have constructed telomerase or luciferase gene expressing SB-transposons with a SV40 enhancer (pT3.hTERT.Con and pT3.Con, respectively) or without an enhancer (pT3.Pro). Using the SB transposon system in vitro hTERT gene overexpression has protective effects from acute cellular injury by tert-butyl hydroperoxide (t-BH), carbon tetrachloride (CCl4), and D-galactosamine (D-GaIN) in normal human cells IMR-90. pT3.hTERT.Con vector and helper plasmid co-transfection resulted in a similar to 3-fold increase in telornerase activity which was maintained for 14 days. Trypan blue and Cell Death Detection Assays showed the protective effects of the telomerase gene against toxic agents. Fourteen days after co-transfection with pT3.hTERT.Con vector and helper plasmid, IMR-90 cells were incubated with 1.2 mM t-BH for 50 min, 5 mM CCl4 for 1.5 h or 30 MM D-GaIN for 24 h. Cell viability of SB-mediated telomerase overexpressing cells significantly increased by 48% (t-BH), 43% (CCl4), and 25% (D-GaIN) in comparison to mock treated cells. Cell Death Detection ELISA showed a decrease in the rate of apoptosis by 47%. In summary, SB transposon mediated telomerase gene transfer may have a protective effect against t-BH, CCl4, or D-GaIN induced acute cellular injury, and this results suggested SB-mediated telomerase therapy for tissue engineering. (C) 2007 Elsevier Inc. All rights reserved.